1. Field of the Invention
The present invention relates generally to the fields of immunology and molecular biology. The invention relates to vectors useful for the expression of immunoglobulin-like domains and fragments, such as T-cell variable domains and wild type and engineered antibody Fc fragments; to bacterial cells capable of secreting said domains, engineered domains, heterodimers and fusion proteins in large quantities; to recombinant domains, fusion proteins or other engineered proteins which have modified in vivo stability; and to methods for their production and use.
2. Description of Related Art
Most glycoproteins that mediate antigen recognition or cell--cell recognition in the immune system contain related structural elements, suggesting that they share a common evolutionary history. Such genes and the resultant protein elements are thus members of an evolutionary conserved group, which has been termed the immunoglobulin (Ig) superfamily. Included in the immune system branch of the immunoglobulin superfamily are antibodies, T cell receptors, MHC glycoproteins, the CD2, CD4 and CD8 proteins, some of the polypeptide chains of the CD3 complex associated with the T cell receptor, various Fc receptors on lymphocytes and other white blood cells, and .beta..sub.2 -microglobulin.
All of the above molecules contain one or more immunoglobulin (Ig)-like domains which exhibit considerable structural homology. Each immunoglobulin-like domain typically has about 100 amino acids and is thought to be folded into a characteristic structure with two antiparallel .beta. sheets, usually stabilized by a conserved disulfide bond. Many of these molecules are dimers or higher oligomers in which Ig homology units of one chain interact with those in another. Other key molecules which contain immunoglobulin-like domains have also been identified, these include receptors such as the PDGF receptor, and cell adhesion molecules such as N-CAM and Ng-CAM.
The majority of T cells recognize antigenic peptides bound to class I or II proteins of the major histocompatibility complex (MHC) and are thus "MHC restricted". The recognition of peptide-MHC complexes is mediated by surface-bound T cell receptors (TCRs). These receptors are comprised of various heterodimeric polypeptides, the majority of which are .alpha. and .beta. polypeptides. A minor population (1-10%) of mature T-cells bears T-cell receptors (TCRs) comprising .delta. .gamma. heterodimers (Borst et al., 1987; Brenner et al., 1986).
The production of single or heterodimeric T-cell receptor variable domains is of interest for purposes of studying T-cell receptor interaction with antigens and developing approaches to therapies for autoimmune diseases and cancer. An important goal of molecular biology is a detailed understanding at the molecular level of the binding of T-cell receptors to cognate peptide-major histocompatibility complexes. Detailed knowledge of this process will be a step in the development of, for example, immunotherapy for T-cell mediated autoimmune disease. Despite this interest and the potential applications arising from the study of T-cell receptor domains, no methods are available for the production of only single T-cell receptor domains, nor has expression and secretion in prokaryotic hosts been successful.
Several composite dimeric species incorporating the .alpha. and .beta. polypeptides have been produced in various systems. TCR .alpha..beta. heterodimers have been expressed as phosphatidyl-inositol linked polypeptides (Lin et al., 1990) or TCR-immunoglobulin chimeras (Gregoire et al., 1991) in mammalian transfectomas. The production of V.sub..alpha. C.kappa. homodimers (Mariuzza & Winter, 1989) and V.beta.-C.beta. monomers (Gascoigne, 1990) in mammalian cells has also been described. The expression and secretion of immunoglobulin VH domains (Ward et al., 1989), Fv fragments (Skerra and Pluckthum, 1988; Ward et al., 1989) and Fab fragments (Better et al., 1988) has been reported. Molecular modeling analyses indicate that there are structural similarities between immunoglobulin F.sub.ab fragments and the extracellular domains of TCRs (Novotny et al., 1986; Chothia et al., 1988). Several expression systems for the production of recombinant TCRs in mammalian cell transfectomas have been documented but successful expression and secretion of these proteins in a prokaryotic host has not been reported.
Despite apparent expression of a single chain anti-fluorescein TCR in E. coli (Novotny et al., 1991), the product could not be isolated from the periplasm even though the leader sequence had been cleaved from the N-terminus of the recombinant protein. The single chain TCR was relatively insoluble, requiring the use of genetic manipulation to replace five of the "exposed" hydrophobic residues with relatively hydrophilic residues.
No methods are presently available for the production of single or heterodimeric T-cell receptor variable domains as secreted proteins. If available, such species would have potential use in the induction of antibodies as protective vaccines, for the therapy of autoimmune disease, and antibodies for targeting idiotypes (T-cell) or T-cell leukemias. Additionally, secretion of T-cell receptor domains from bacterial cell hosts should provide a convenient, economically attractive and rapid route for production of recombinant T-cell receptors.
Immunoglobulin Fc domains are also of great interest for purposes of studying the mechanisms of antibody stabilization, catabolism and antibody interactions with further molecules of the immune system. These include, depending on the class of antibody, interactions with complement, and binding to specific receptors on other cells, including macrophages, neutrophils and mast cells. More detailed knowledge of the biology of Fc regions would be important in understanding various molecular processes of the immune system, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and allergic reactions.
In an analogous situation to the T cell receptor, methods do not currently exist which allow the production of large quantities of functional individual Fc domains. To date, it is not known whether an immunoglobulin fragment, let alone a stable one, could be produced in a prokaryotic cells such as an E. coli. Therefore, methods for the recombinant production of such domains in prokaryotic cells, and particularly, methods resulting in the secretion of large quantities of antibody constant domains would be advantageous.
The production and purification of Fc domains would allow their structural and functional properties to be more precisely defined, and ultimately, precise interactive residues identified which control various functions, such as, e.g., catabolism. The production of a stable Fc fragment that has reduced binding to Fc receptors and complement factors would be attractive, since such a fragment could be used to tag therapeutic reagents. Chimaeric proteins produced in this manner would have the advantage of high stability and in addition, would not bind significantly to Fc receptors that are expressed on a wide variety of immune cells (Hogg, 1988). This would reduce the non-specific binding of the therapeutic reagents, since Fc receptors are ubiquitously expressed. This, in turn, allows lower doses of the agent to be used in therapy and also reduces undesirable side-effects due to non-specific binding.
TCR fragments have been produced in mammalian cells but they are relatively large. Whole antibodies have also been produced recombinantly. However, the availability of smaller size immunoglobulin-like segments may allow more rapid structural resolution using such techniques as NMR and X-ray crystallography. TCR variable domains interact with peptide-MHC complexes and are of considerable interest, antibody constant domains are also of interest given their wide variety of functions. The availability of purified individual domains will allow the functions of residues within such proteins to be defined.
Additionally, the use of variable domains alone in immunization should result in the production of anti-variable domain antibodies. Such antibodies are expected to be particularly desirable for use in therapy and diagnosis since they block the interaction of the TCR with antigen and, due to the variable nature of the V.sub..alpha. /V.sub..beta. domains or other domains such as V.sub..delta. and V.sub..gamma., are specific for subsets of T-cells. Large TCR fragments, such as those that can be expressed from mammalian cells, result in production of antibodies not only against the variable domains, but also against other domains present in the construct, such as TCR constant domains and/or other immunoglobulin domains. There would therefore be distinct advantages in having smaller variable domain TCR fragments available, particularly for immunization since any immune response generated is likely to be directed to particular regions of interest, i.e., the V domains.